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Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 399-411 ISSN: 2319-7706 Volume 4 Number 2 (2015) pp. 399-411 http://www.ijcmas.com Original Research Article The Sensitivity of Escherichia coli to Extracts of Combretum fragrans, Combretum micranthum and Combretum molle Locally Used in the Treatment of Diarrheal Diseases in the Far-North Region of Cameroon Toua Vroumsia1*, Pierre Saotoing1, Amadou Dawé2, Moussa Djaouda1, Mbende Ekaney1 and Beltin Abeng Mua1 1Department of Life and Earth Sciences, The University of Maroua Higher Teachers Training College, P. O. Box 55 Maroua, Cameroon 2Department of Chemistry, The University of Maroua Higher Teachers Training College, P. O. Box 55 Maroua, Cameroon *Corresponding author A B S T R A C T This study was undertaken to determine the sensitivity of Escherichia coli culture to extracts of three locally used antibacterial plants (Combretum molle, Combretum fragrans and Combretum micranthum) and to identify the phytochemicals present in these plants. Acetone was used as the solvent for the extraction of the leaves. K e y w o r d s The sensitivity of Escherichia coli culture to the plants extracts was determined on Mueller Hinton Agar, following the agar well diffusion technique. Acetone was Combretum, used as a negative control and Ciprofloxacin as positive control. The Minimum Plant extracts, Inhibitory Concentration of the plant extracts was determined by a modified broth Sensitivity, dilution method. Phytochemical test was done by detecting major colour changes. Escherichia coli, Results indicated that all tested plants extracts, as well as Ciprofloxacin, Inhibition zone, demonstrated antibacterial activity, with diameters of inhibition zones ranging from Minimum 8 ± 0.35 to 36 ± 1.80 mm, while acetone had no activity. Combretum molle and C. Inhibitory fragrans had the greatest inhibitory activity against E. coli. The found MIC varied Concentration from 0.625 to 2.50 mg/ml. Phytochemical tests showed that the plants contained tannins, proteins, flavonoids, phenols, coumarines and glycosides. These results justify the use of the tested plants as antibacterial by the population. Further studies, involving the plants extracts antimicrobial activities against other micro- organisms and their in vivo toxicity and mechanism of action are needed. Introduction Diarrhoeal diseases are one of the foremost diarrhoea is prevalent in developing public health problems worldwide. In the countries, particularly in tropical regions 21st century, diarrhoeal diseases continue to (Pickering, 2004). Enteric pathogens are the be a major cause of morbidity and mortality most frequent causes of diarrhoea illness, worldwide (O Ryan et al., 2005). Recurrent which account for an annual mortality rate 399 Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 399-411 of 3 million and an estimated 4 billion reason why scientific research in the field of infections worldwide (Talaro, 2005). Almost traditional medicinal plants has to be half of the world s population suffer from intensified in order to find alternative diseases associated with insufficient or antimicrobial drugs. Numerous studies have contaminated water and is at risk from identified active compounds within herbal waterborne and foodborne diseases, of plants that are effective antibiotics (Basile et which diarrheal diseases are the most deadly al., 2000) and some traditional remedies (Sindiga et al., 1995). have already produced compounds that are effective against antibiotic-resistant strains The pathogenic bacteria most commonly of bacteria (Koné et al., 2004). associated with endemic forms of diarrhoea are diarrheagenic Escherichia coli, The genus Combretum includes almost 400 Salmonella spp, Shigella, Vibrio cholerae, species found all across Africa, many of Aeromonas and Pleisomonas spp (Mamatha, which are widely used in African traditional 2006). Diarrhoea is also caused by other medicine (Van Wyk and Gericke, 2000). agents like viruses and parasites (Palombo, Several species of the genus have been 2006). reported for their biological activities. Different extracts (ethanol, chloroform, Antibiotic resistance is a major clinical methanol or water) of C. micranthum cause of concern in treating infections antibacterial activity against a number of caused by microorganisms (Acharyya et al., microorganisms such as Pseudomonas 2009). For instance, E. coli has showed aeruginosa, Staphylococcus aureus, resistance to the common cheap antibiotics, Salmonella species, Streptococcus species, notably Trimethoprim-Sulphamethoxazole, Proteus vulgaris, Klebsiella species, Sarcina Kanamycin and Gentamycin (Bebora et al., lutea, Micrococcus luteus and Bacillus 1994). As resistance to old antibiotics subtilis was noted (Neuwinger, 2000). spreads, the development of new Antifungal activity against Candida antimicrobial agents has to be expedited if albicans, antiviral activity against Herpes the problem is to be contained. However, the simplex 1 and 2, antimalarial activity against past record of rapid, wide spread emergence Plasmodium falciparum and antidiabetic of resistance to newly introduced activity was also reported (Masoko et al., antimicrobial agents indicates that even new 2007). Extracts of C. erythrophyllum have families of antimicrobial agents will have a shown antibacterial activity at different short life expectancy (Coates et al., 2002). doses against E. coli, Pseudomonas aeruginosa, Staphylococcus aureus and Medicinal plants are effective in the Enterococcus faecalis (Martini and Eloff, treatment of infectious diseases, while 1998). simultaneously mitigating many of the side effects that are often associated with Phytochemical studies carried out in the synthetic drugs (Koné et al., 2004). Multiple genus Combretum have demonstrated the drug resistance has developed due to the occurrence of many classes of constituents, indiscriminate use of synthetic antimicrobial primarily terpenoids (mainly triterpenes) and drugs (Davis, 1994). In addition, bacteria phenolic compounds (flavonoids, have evolved numerous defences against the stilbenoids, phenanthrenes), among others antimicrobials (Ahmad and Aqil, 2006). A (de Morais Lima et al., 2012). combination of these factors is the more 400 Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 399-411 As the plants of Combretum genus are they were ground into powder form using a locally and widely used in the treatment of grinder (MK 10-525-B). diarrheal diseases in the Far-North region of Cameroon, it becomes necessary to verify Extraction the scientific basis of this use and to have an idea on the phytochemical composition of All chemicals used in this study were the plants. purchased from Sigma-Aldrich chemicals Co (St. Louis, MO, USA) and were of The general objective of this study is to analytic reagent grade. The powdered plant evaluate the sensitivity of E. coli to extracts materials were soaked in acetone for 48 of the leaves of Combretum molle, hours with intermittent shaking to allow the Combretum fragrans and Combretum active phytochemicals to dislodge in the micranthum. To attempt this, the following solvent. The acetone soaked plant extracts specific objectives are focused: investigation were then filtered by the use of a Whatman of E. coli susceptibility to extracts of the No.1 filter paper and the filtrates evaporated plants leaves, finding of the minimum by using a rotary evaporator (W.2000 inhibitory concentration of the leaves Heidolph, Germany) set at 72oC to remove extracts and screening of the leaves extracts, the excess solvent. The extracts were stored in order to identify the phytochemical in air tight containers in the laboratory until compounds present in these extracts. when they were used. Materials and methods Antimicrobial assays Description of the study area Test cultures The Far-North region is the northern most Bacteria strain of E. coli was isolated from constituent region of the Republic of human faecal matter from patients who Cameroon. It borders the North region to the presented diarrhoea at the Garoua Annex of south, Chad to the east and Nigeria to the the Pasteur Centre of Cameroon. This o west. It is located on latitude 11°00N and bacteria isolate was maintained at 4 C on longitude 14°30E and has a Sudanno- nutrient agar slants until when it was used. sahalian climate, with steppes and savannah vegetation. The annual rainfall is about 400- Preparation of the Mueller Hinton Agar 1500 mm and the annual average (MHA) culture Medium temperature is 28°C. The preparation was done following the Collection of plants materials manufacturer instructions. Generally, 7.6 g of the MHA was added to 200 ml of distilled Fresh plant leaves of Combretum molle R. water and was brought to boil using a Br. ex G. Don, Combretum fragrans F. Bunsen burner. The medium was, later on, Hoffm. and Combretum micranthum G. Don sterilized by autoclaving at 1210C for 15 were harvested in Ngassa, a small village minutes. It was then removed and placed in about a few kilometres from Mindif, in the Petri dishes in a homogenous manner and Far-North Region of Cameroon. The plant allowed to solidify. The Petri dishes leaves were washed and dried at ambient containing agar were then stored in a temperature under shade after collection refrigerator to be used the following day. until they were dry. For laboratory analysis, 401 Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 399-411 Determination of diameters of growth The medium was then poured into sterile inhibition zones Petri dishes and allowed to set. The surface of the medium was